Protein activity as a function of interaction

A protein's biological activities are dictated by its interactions with other proteins. Protein-protein interactions control cellular processes including protein modification, transport, folding, signaling, and cell cycling. In order to fully understand protein function, proteins should be studied in the context of their interactions with other proteins.

MS techniques used for protein-protein interaction

Native mass spectrometry (MS) plays a crucial role in generating information on protein-protein interactions, confirming protein identity, and elucidating heterogeneity data. Solvent manipulation during protein complex introduction into the mass spectrometer can reveal additional information. For example, varying solvent pH or adding organic additives can separate protein subcomplexes, which are then fragmented to confirm identity. Native MS can also give stoichiometry information on the proteins involved in protein-protein interactions.

Crosslinking mass spectrometry

In crosslinking mass spectrometry (XL-MS), chemical crosslinkers are used to join components of interacting complexes in order to maintain their original interaction. A bottom-up proteomics MS approach is then used. One advantage of XL-MS is that only a small sample size is required, with analysis performed directly at the proteome level. XL-MS also enables protein interactions to be examined at the physiological state of an organism, generating information that is biologically relevant.

Hydrogen deuterium exchange mass spectrometry

Hydrogen deuterium exchange mass spectrometry (HDX-MS) helps localize protein-protein interaction sites. Typically, areas of protein-protein interaction have hindered exchange rates between amide protons and D2O compared to regions with direct exposure to D2O. Because of this difference, protein-protein interaction sites can be localized using proton exchange. MS then identifies those sites. HDX experiments are usually performed on proteins using conditions that are native to their biological state.

Featured video

Fan Liu, PhD, of Utrecht University, describes the benefits of crosslinking mass spectrometry and how the XlinkX software enables analysts to search any protein database, up to proteome-level size.